MC4R mutations represent the largest monogenic cause of obesity, resulting mainly from receptor misfolding and intracellular retention by the cellular quality control system. The present study aimed at determining whether pharmacological chaperones (PC) that restore folding and plasma membrane trafficking by stabilizing near native protein conformation, may represent valid therapeutic avenues for the treatment of melanocortin type 4 receptor (MC4R) linked obesity.To test the therapeutic PC potential, we engineered humanized MC4R mouse models expressing either the wild type (WT) human MC4R or a prevalent obesity-causing mutant (R165W). Administration of a PC able to rescue cell surface expression and functional activity of R165W-hMC4R in cells, restored the anorexigenic response of the R165W-hMC4R obese mice to melanocortin agonist, providing a proof-of-principle for the therapeutic potential of MC4R-targetting PC in vivo. Interestingly, the expression of the WT-hMC4R in mice revealed lower sensitivity of the human receptor to alpha-melanocyte-stimulating hormone (α-MSH) but not β-MSH or MTII, resulting in a lower penetrance obese phenotype in the WT-hMC4R versus R165W-hMC4R mice. In conclusion, we created two new obesity models, one hypomorph highlighting species differences, and one amorphic that provides a pre-clinical model to test the therapeutic potential of PC to treat MC4R-linked obesity.
Patricia René, Damien Lanfray, Denis Richard, Michel Bouvier
Reestablishing an appropriate balance between T effector cells (Teff) and T regulatory cells (Treg) is essential for correcting autoimmunity. Multiple Sclerosis (MS) is an immune-mediated chronic central nervous system (CNS) disease characterized by neuroinflammation, demyelination, and neuronal degeneration, in which the Teff:Treg balance is skewed toward pathogenic Teff cells, Th1 and Th17 cells. Signal transducer and activator of transcription 3 (STAT3) is a key regulator of Teff:Treg balance. Using the structure-based design, we have developed a novel small-molecule prodrug LLL12b that specifically inhibits STAT3 and suppresses Th17 differentiation and expansion. Moreover, LLL12b regulates the fate decision between Th17 and Tregs in an inflammatory environment, shifting Th17:Treg balance toward Tregs and favoring the resolution of inflammation. Therapeutic administration of LLL12b after disease onset significantly suppresses disease progression in adoptively transferred, chronic, and relapsing-remitting experimental autoimmune encephalomyelitis. Disease relapses were also significantly suppressed by LLL12b given during the remission phase. Additionally, LLL12b shifts Th17:Treg balance of CD4 T cells from MS patients toward Tregs and increases Teff sensitivity to Treg-mediated suppression. These data suggest selective inhibition of STAT3 by the novel small molecule LLL12b recalibrates the effector and regulatory arms of CD4 T responses, representing a potentially clinically translatable therapeutic strategy for MS.
Saba I. Aqel, Xiaozhi Yang, Emma E. Kraus, Jinhua Song, Marissa F. Farinas, Erin Y. Zhao, Wei Pei, Amy E. Lovett-Racke, Michael K. Racke, Chenglong Li, Yuhong Yang
Diarrhea is a major cause of global mortality, and outbreaks of secretory diarrhea such as cholera remain an important problem in the developing world. Current treatment of secretory diarrhea primarily involves supportive measures such as fluid replacement. The calcium-sensing receptor (CaSR) regulates multiple biological activities in response to changes in extracellular Ca+2. The FDA-approved drug cinacalcet is an allosteric activator of CaSR used for treatment of hyperparathyroidism. Here, we found by short-circuit current measurements in human colonic T84 cells that CaSR activation by cinacalcet reduced forskolin-induced Cl- secretion by greater than 80%. Cinacalcet also reduced Cl- secretion induced by cholera toxin, heat-stable E. coli enterotoxin, and vasoactive intestinal peptide (VIP). The cinacalcet effect primarily involved indirect inhibition of cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl- secretion following activation of CaSR, and downstream phospholipase C and phosphodiesterases. In mice, cinacalcet reduced fluid accumulation by more than 60% in intestinal closed-loop models of cholera and Traveler’s diarrhea. The cinacalcet effect involved both inhibition of CFTR-mediated secretion and stimulation of sodium-hydrogen exchanger 3 (NHE3)-mediated absorption. These findings support the therapeutic utility of the safe and commonly used drug cinacalcet in CFTR-dependent secretory diarrheas including cholera, Traveler’s diarrhea and VIPoma.
Apurva A. Oak, Parth D. Chhetri, Amber Rivera, Alan S. Verkman, Onur Cil
Osteosarcoma (OS) is an aggressive mesenchymal tumor for which no molecularly targeted therapies are available. We have previously identified TRAF2 and NCK-interacting protein kinase (TNIK) as an essential factor for the transactivation of Wnt signal target genes and shown that its inhibition leads to eradication of colorectal cancer stem cells. The involvement of Wnt signaling in the pathogenesis of OS has been implicated. The aim of the present study was to examine the potential of TNIK as a therapeutic target in OS. RNA interference or pharmacological inhibition of TNIK suppressed the proliferation of OS cells. Transcriptome analysis suggested that a small-molecule inhibitor of TNIK up-regulated the expression of genes involved in OS cell metabolism and down-regulated transcription factors essential for maintaining the stem cell phenotype. Metabolome analysis revealed that this TNIK inhibitor redirected the metabolic network from carbon flux towards lipid accumulation in OS cells. Using in vitro and in vivo OS models, we confirmed that TNIK inhibition abrogated the OS stem cell phenotype, simultaneously driving conversion of OS cells to adipocyte-like cells through induction of peroxisome proliferator-activated receptor-γ. In relation to potential therapeutic targeting in clinical practice, TNIK was confirmed to be in an active state in OS cell lines and clinical specimens. From these findings, we conclude that TNIK is applicable as a potential target for treatment of OS, affecting cell fate determination.
Toru Hirozane, Mari Masuda, Teppei Sugano, Tetsuya Sekita, Naoko Goto, Toru Aoyama, Takato Sakagami, Yuko Uno, Hideki Moriyama, Masaaki Sawa, Naofumi Asano, Masaya Nakamura, Morio Matsumoto, Robert Nakayama, Tadashi Kondo, Akira Kawai, Eisuke Kobayashi, Tesshi Yamada
Management of Gastrointestinal stromal tumor (GIST) has been revolutionized by the identification of activating mutations in KIT and PDGFRA and clinical application of receptor tyrosine kinase (RTK) inhibitors in advanced disease. Stratification of GIST into molecularly defined subsets provides insight into clinical behavior and response to approved targeted therapies. Although these RTK inhibitors are effective in most GIST, resistance remains a significant clinical problem. Development of effective treatment strategies for refractory GIST requires identification of novel targets to provide additional therapeutic options. Global kinome profiling has potential to identify critical signaling networks and reveal protein kinases essential in GIST. Using Multiplexed Inhibitor Beads and Mass Spectrometry, we explored the majority of the kinome in GIST specimens from the three most common molecular subtypes (KIT-mutant, PDGFRA-mutant, Succinate dehydrogenase (SDH)-deficient) to identify novel kinase targets. Kinome profiling with loss-of-function assays identified an important role for G2-M tyrosine kinase, Wee1, in GIST cell survival. In vitro and in vivo studies revealed significant efficacy of MK-1775 (Wee1 inhibitor) in combination with avapritinib in KIT and PDGFRA-mutant GIST cell lines, and notable efficacy of MK-1775 as a monotherapy in the PDGFRA-mutant line. These studies provide strong preclinical justification for the use of MK-1775 in GIST.
Shuai Ye, Dinara Sharipova, Marya Kozinova, Lillian R. Klug, Jimson W. D'Souza, Martin G. Belinsky, Katherine J. Johnson, Margret B. Einarson, Karthik Devarajan, Yan Zhou, Samuel Litwin, Michael C. Heinrich, Ronald P. DeMatteo, Margaret von Mehren, James S. Duncan, Lori Rink
In this work, we have explored natural unmodified low- and high-density lipoproteins (LDL and HDL) as selective delivery vectors in colorectal cancer therapy. We show in vitro in cultured cells and in vivo (NanoSPECT/CT) in the CT-26 mice colorectal cancer model that LDLs are mainly taken up by cancer cells, while HDLs are preferentially taken up by macrophages. We loaded LDLs with cisplatin and HDLs with the heat shock protein-70 inhibitor AC1LINNC, turning them into a pair of “Trojan horses” delivering drugs selectively to their target cells as demonstrated in vitro in human colorectal cancer cells and macrophages, and in vivo. Coupling of the drugs to lipoproteins and stability was assessed by mass and raman spectrometry analysis. Cisplatin vectorized in LDLs led to better tumor growth suppression with strongly reduced adverse effects such as a renal or liver toxicity. AC1LINNC vectorized into HDLs induced a strong oxidative burst in macrophages and innate anti-cancer immune response. Cumulative anti-tumor effect was observed for both drug-loaded lipoproteins. Altogether, our data show that lipoproteins from patient’s blood can be used as natural nanocarriers allowing cell specific targeting, paving the way toward more efficient, safer and personalized use of chemo-and immunotherapeutic drugs in cancer.
Tarik Hadi, Christophe Ramseyer, Thomas Gautier, Pierre-Simon Bellaye, Tatiana Lopez, Antonin Schmitt, Foley Sarah, Semen Yesylevskyy, Thibault Minervini, Romain Douhard, Lucile Dondaine, Lil Proukhnitzky, Samir Messaoudi, Maeva Wendremaire, Mathieu Moreau, Fabrice Neiers, Bertrand Collin, Franck Denat, Laurent Lagrost, Carmen Garrido, Frederic Lirussi
Adoptive cell therapy involves the infusion of tumor-reactive T cells into patients with cancer to provide antitumor immunity. The ex vivo expansion and differentiation of such T cells are key parameters that affect their therapeutic potential. Human T cells are presently expanded in culture through the use of anti-CD3 and anti-CD28 mAbs immobilized on beads, expressed on cells, or assembled in the context of soluble antibody complexes. Here we report the design of a small, bispecific single-chain variable fragment construct agonizing both CD3 and CD28 pathways. This soluble T cell expansion protein, termed T-CEP, activates, expands, and differentiates human T cells ex vivo at concentrations in the femtomolar range. Importantly, T-CEP promotes the preferential growth of human CD8+ T cells over the course of 12 days in comparison with methods involving immobilized anti-CD3 mAb/soluble anti-CD28 mAb or soluble anti-CD3/CD28 mAb complexes. The differentiation profile of the resulting human T cell population is also singularly affected by T-CEP, favoring the expansion of a preferred CD8+CD27+ T cell phenotype. The activity profile of T-CEP on human T cells ex vivo suggests its use in generating human T cell populations that are more suited for adoptive cell therapy.
Esther I. Matus, Amanda Sparkes, Jean Gariépy
Chimeric antigen receptor (CAR) T cell therapy for solid tumors has shown limited efficacy in early-phase clinical studies. The majority of CARs encode CD28 and/or 41BB costimulatory endodomains, and we explored whether MyD88 and CD40 (MC) costimulatory endodomains in CARs could improve their antitumor activity. We generated CD28-, 41BB-, and MC-CAR T cells and demonstrated that MC-CAR T cells have greater proliferative capacity and antitumor activity in repeat stimulation assays and in tumor models in vivo. Transcriptomic analysis revealed that MC-CAR T cells expressed higher levels of MYB and FOXM1, key cell cycle regulators, and were activated at baseline. After stimulation, MC-CAR T cells remained in a less differentiated state than CD28- and 41BB-CAR T cells as judged by low levels of transcription factor TBET and B lymphocyte induced maturation protein 1 expression and lower cytolytic activity in comparison with CD28- and 41BB-CAR T cells. Thus, including MyD88 and CD40 signaling domains in CARs may improve current CAR T cell therapy approaches for solid tumors.
Brooke Prinzing, Patrick Schreiner, Matthew Bell, Yiping Fan, Giedre Krenciute, Stephen Gottschalk
We remain largely without effective prophylactic/therapeutic interventions for COVID-19. Although many human COVID-19 clinical trials are ongoing, there remains a deficiency of supportive preclinical drug efficacy studies to help guide decisions. Here we assessed the prophylactic/therapeutic efficacy of hydroxychloroquine (HCQ), a drug of interest for COVID-19 management, in two animal disease models. The standard human malaria HCQ prophylaxis (6.5 mg/kg given weekly) and treatment (6.5 mg/kg) did not significantly benefit clinical outcome nor reduce SARS-CoV-2 replication/shedding in the upper and lower respiratory tract in the rhesus macaque disease model. Similarly, when used for prophylaxis or treatment neither the standard human malaria dose (6.5 mg/kg) nor a high dose (50 mg/kg) of HCQ had any beneficial effect on clinical disease or SARS-CoV-2 kinetics (replication/shedding) in the Syrian hamster disease model. Results from these two preclinical animal models may prove helpful in guiding clinical use of HCQ for prophylaxis/treatment of COVID-19.
Kyle Rosenke, Michael A. Jarvis, Friederike Feldmann, Benjamin Schwarz, Atsushi Okumura, Jamie Lovaglio, Greg Saturday, Patrick W. Hanley, Kimberly Meade-White, Brandi N. Williamson, Frederick A. Hansen, Lizette Pérez-Pérez, Shanna Leventhal, Tsing-Lee Tang-Huau, Julie Callison, Elaine Haddock, Kaitlin A. Stromberg, Graham Sewell, Dana Scott, Catharine M. Bosio, David W. Hawman, Emmie de Wit, Heinz Feldmann
Infections caused by multi-drug resistant Staphylococcus aureus, especially MRSA, are responsible for high mortality and morbidity worldwide. Resistant lineages were previously confined to hospitals, but are now also causing infections among healthy individuals in the community. It is therefore imperative to explore therapeutic avenues that are less prone to raise drug resistance compared to today’s antibiotics. An opportunity to achieve this ambitious goal could be provided by targeted antimicrobial photodynamic therapy (aPDT), which relies on the combination of a bacteria-specific targeting agent and light-induced generation of reactive oxygen species by an appropriate photosensitizer. Here we conjugated the near-infrared photosensitizer IRDye700DX to a fully human monoclonal antibody, specific for the invariantly expressed staphylococcal antigen IsaA. The resulting immunoconjugate 1D9-700DX was characterized biochemically and in preclinical infection models. As demonstrated in vitro, in vivo, and in a human post-mortem orthopedic implant infection model, targeted aPDT with 1D9-700DX is highly effective. Importantly, combined with the non-toxic aPDT-enhancing agent potassium iodide, 1D9-700DX overcomes the antioxidant properties of human plasma and fully eradicates high titers of MRSA. We show that the developed immunoconjugate 1D9-700DX targets MRSA and kills it upon illumination with red light, without causing collateral damage to human cells.
Mafalda Bispo, Andrea Anaya-Sanchez, Sabrina Suhani, Elisa J.M. Raineri, Marina López-Álvarez, Marjolein Heuker, Wiktor Szymański, Francisco Romero Pastrana, Girbe Buist, Alexander R. Horswill, Kevin P. Francis, Gooitzen M. van Dam, Marleen van Oosten, Jan Maarten van Dijl
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